An innovative dual recognition aptasensor for specific detection of Staphylococcus aureus based on Au/Fe3O4 binary hybrid

Pathogenic bacteria cause disease outbreaks and threaten human health, prompting the research on advanced detection assays. Herein, we developed a selective molecular imprinted aptasensor for sensitive and prompt quantitation of Staphylococcus aureus (S. aureus) bacteria. The aptasensor was constructed by immobilization of aptamer on gold nanoparticles modified magnetic nanoparticles (apt-AuNPs@ Fe3O4). A functional monomer (o-phenylenediamine, o-phen) was electro-polymerized on the surface of the as-synthesized nanocomposite in the presence of a template (S. aureus). After removing S. aureus, the formed imprinted sites were available to extract pathogenic bacteria from complicated matrices. The surface morphology of the as-fabricated nanocomposites was characterized using different spectroscopic and electrochemical methods. Moreover, we thoroughly evaluated factors affecting the synthesis and determination procedures. The molecular imprinted aptasensor exhibited a wide linear range of 101–107 CFU mL−1 with a Limit of Detection, LOD (signal to noise = 3) of 1 CFU mL−1. The aptasensor detected S. aureus in milk, conduit water, and apple juice samples with good recoveries % and satisfactory relative standard deviations (RSDs %) values.


Fabrication of MIP-apt-AuNPs@Fe 3 O 4 /GCE.
.0 μL of the S. aureus-apt complex (prepared in "Activation and preparation of aptamer (apt)") was dropped on the surface of the AuNPs/Fe 3 O 4 /GCE where apt was covalently bound to AuNPs by strong Au-S bond. Then, Then, 15 μL of the S. aureus was cast on the electrode's surface to impregnate any free apt. Secondly, 1.45 mM o-phen was electro-polymerized on the surface of the electrode by sweeping the potential in the range of -0.4-0.9 V using a scan rate 100 mV s −1 for 15 cycles. Finally, the electrode was placed in a solution containing 0.01 M SDS and 7% HNO 3 (dissolved in DDW) for 60 min to remove S. aureus from its imprinted sites. A non-imprinted polymer (NIP) was prepared using the same steps without adding the template (S. aureus) (Fig. 1).
Preparation of real samples. The milk sample (0.5 mL) was mixed with 1.5 mL acetonitrile and spiked with S. aureus before shaking for 30 s. The mixture was sonicated for 5 min and then centrifuged at 3000 rpm for 10 min, and the supernatant was collected for further analysis. Drug-free milk samples were prepared using the same steps without spiking with S. aureus 26 . Conduit water was filtered to remove the insoluble and floated matters and stored in high-quality clean polyethylene containers. Water samples (5.0 mL) were spiked with different concentrations of S. aureus and stored at − 4 °C until analysis 22,27,28 . Apple juice samples obtained from the local market were analyzed without any further treatment.

Results and discussions
Characterization of nanocomposites. Scanning Electron Microscope (SEM) was used to check the different synthesized nanomaterials as depicted in Fig.S1. Magnetic nanoparticles (Fe 3 O 4 NPs) are uniformly distributed with an average size of 14.5 nm (Fig. S1A). After modification with AuNPs, the size was increased to 38.5 nm, suggesting the successful decoration of Fe 3 O 4 NPs with AuNPs (Fig. S1B). Functional monomer (o-phen) was electro-polymerized on the surface of AuNPs@Fe 3 O 4 /GCE in the presence of template (S. aureus) and thiolated apt, resulting in the complete coverage with a film of o-phen polymer (NIP) (Fig. S1C). After the removal of S. aureus from its imprinted sites, narrow pores were formed on the surface of polymer network resulting in the formation of molecular imprinted polymer (MIP) film (Fig. S1D). Fig.S2 shows the energy dispersive X-ray spectroscopy (EDX) of AuNPs@Fe 3 O 4 nanocomposite with the main elements of O, Fe, and Au. Electrochemical characterization of the as-synthesized nanocomposites. Different electrodes were prepared and evaluated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). They were immersed in a solution of 5.0 mM Fe(CN) 6 3-/4dissolved in 0.1 M KCl. Figure 2A a exhibits the redox peaks of Fe(CN) 6 3-/4at bare GCE where it shows to identifiable and separated anodic and cathodic peaks. After modification with Fe 3 O 4 NPs, the redox currents of the redox probe were increased as a result of enhanced www.nature.com/scientificreports/    (Fig. 2Ab). Further enhancement of the redox currents was observed after modification with AuNPs ( Fig. 2Ac), suggesting the excellent conductivity of AuNPs. Attachment of apt to the surface of AuNPs@ Fe 3 O 4 /GCE resulted in the decrease of the peak currents of Fe(CN) 6 3-/4due to the repulsion between the negatively charged apt and negatively charged redox probe (Fig. 2Ad). Electropolymerization of o-phen monomer on the surface of apt-AuNPs@ Fe 3 O 4 /GCE and in the presence of S. aureus i.e. NIP-apt-AuNPs@ Fe 3 O 4 /GCE, the peak currents of Fe(CN) 6 3-/4were sharply deceased due to the formation of insulating layer that inhibited the influx of the redox probe (Fig. 2Ae). The removal of S. aureus from its imprinted sites i.e. MIP-apt-AuNPs@ Fe 3 O 4 /GCE increased the peak currents of Fe(CN) 6 3-/4due to the creation of numerous imprinted sites for the flowing of the redox probe, but it is still lower than apt-AuNPs@ Fe 3 O 4 / GCE (Fig. 2Af). After rebinding the S. aureus, the peak currents of the redox probe were dramatically decreased as the cavities of the imprinted layers were relocked by the template (Fig. 2Ag). Moreover, the electrochemical activities of different interfaces were demonstrated using EIS (Fig. 2B). It is shown that the semicircle diameter was changed after each modification.
Optimization of experimental conditions. Optimizations of incubation time, pH effect, elution time, deposition potential and time of AuNPs, and apt concentration were listed in ESM.
Analytical figures of merit. The sensitivity of the proposed aptasensor towards S. aureus was measured using differential pulse voltammetry (DPV) under optimized conditions. Figure 3A shows that the peak currents of the redox probe at MIP-apt-AuNPs@ Fe 3 O 4 /GCE were decreased after the increase in the concentration of S. aureus. The calibration plot shown in Fig. 3B was linear over the range of 10 1 -10 7 CFU mL -1 with a linear regression of Ipa (µA) = − 23.9 Log C S. aureus + 283.9 (R 2 = 0.9986). According to IUPAC recommendation (IUPAC 1976), the analyte's signal at the detection limit (S dl ) is given by: where S reag is the electrochemical signal for a blank, σreag is the known standard deviation for the blank's electrochemical signal (n = 10). As is well known, k = signal/noise (S/N) = 3. As suggested by Long and Winefordner (1983) (Long and Winefordner 1983), the use of k = 3 allows a confidence level of 99.86% for a normal distribution of the blank signals. The detection limit can be calculated by S dl and calibration curves. The LOD was calculated as 1 CFU mL −1 . Moreover, the method with compared with other reported methods for the determination of S. aureus (Table 1). I t was found that the proposed aptasensor exhibits wide-linear range and low detection value.

Reproducibility, repeatability, and stability of MIP-apt-AuNPs@ Fe 3 O 4 /GCE. Reproducibility
was measured by monitoring the DPV responses of five fabricated aptasensor prepared under the same conditions (Fig. 4A). It was found that the relative standard deviation % (RSD %) did not exceed 3.2%. Moreover, the repeatability was measured via measuring the DPV responses for six readings and calculating the RSD % that did not exceed 2.6% (Fig. 4B).
Moreover, the stability of the aptasensor was examined using CV at scan rate of 300 mV s −1 in 0.1 M phosphate buffer and 5.0 mM [Fe(CN) 6 ] 3−/4− . After 50 cycles, DPV readings did not appreciably change, confirming the excellent stability of the proposed sensor (Fig. S10A). In addition, the long term stability of the aptasensor was studied by storing the aptasensor at 4 °C and it was used to detect S. aureus every week for one month (Fig.S10  B). It was found that the proposed aptasensor retained about 95% of its original activity for four weeks.  Table 2 shows that the recoveries % ranged from 96 to 104% with relative standard deviations (RSDs) less than 3.4%, suggesting the suitability of the aptasensor for measuring S. aureus in milk, conduit water, and apple juice samples. Calibration plots for different artificial samples were shown in Fig. 6.

Conclusion
In this context, an ultrasensitive and selective molecular imprinted based aptasensor was fabricated to detect S. aureus. The aptasensor consists of gold nanoparticles modified magnetic nanoparticles loaded on the glassy carbon electrode surface (AuNPs@ Fe 3 O 4 /GCE). The thiolated aptamer was attached to the nanocomposite surface via Au-S covalent bond. A polymer film was deposited over the surface of the AuNPs@ Fe 3 O 4 /GCE by electro-polymerization of o-phen in the presence of the template (S. aureus). After elution of the template, the formed imprinted cavities were formed that can extract S. aureus from the complicated matrices. Simplicity, low LOD, good stability, low cost, high sensitivity, and high selectivity are the main advantages of the proposed aptasensor. The molecular imprinted aptasensor was applied efficiently for the detection of S. aureus in milk, conduit water, and apple juice samples.

Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files.